Method for the separation and purification of epothilones

Abstract

The invention discloses a method for the separation and purification of epothilones, especially discloses a method for the separation and purification of epothilones B and A using normal phase silica gel chromatography, which comprises loading the sample after dissolving the sample containing epothilones B and A with C 1 -C 7 alkyl halide compounds or mixing the sample with silica gel, then gradient eluting silica gel column by an elution solvent of normal phase silica gel column, and finally obtaining products.

What is claimed is: 
     
       1. A method for separation and purification of epothilones B and A, characterized in that, the separation and purification of epothilones B and A are performed by a normal phase silica gel column chromatography; and the method comprises: dissolving a sample containing epothilones B and A in C 1 -C 7  alkyl halide compound(s) to form a mixture, wherein the mixture is mixed with silica gel or not;
 loading the mixture on a silica gel column; 
 gradient eluting the silica gel column with a normal phase silica gel column eluent; 
 collecting fractions; and 
 obtaining products; 
 wherein the normal phase silica gel column eluent is chosen from a C 1 -C 7  hydrocarbon, a C 1 -C 7  alkyl halide compound, a C 1 -C 7  ketone, C 1 -C 7  ester, or any combination thereof, and wherein the C 1 -C 7  hydrocarbon is chosen from petroleum ether, n-hexane, cyclohexane, n-heptane, or any combination thereof, the C 1 -C 7  alkyl halide compound is chosen from dichloromethane, trichloromethane, bromoethane, or any combination thereof, the C 1 -C 7  ketone is chosen from acetone, butanone, or any combination thereof, and the C 1 -C 7  ester is chosen from ethyl acetate, isobutyl acetate, or any combination thereof; and 
 wherein the normal phase silica gel column is balanced with C 1 -C 7  alkyl halide compound(s) before use. 
 
     
     
       2. The method according to  claim 1 , wherein the method comprises:
 dissolving a sample containing epothilones B and A in C 1 -C 7  alkyl halide compound(s) to form a mixture, wherein the mixture is mixed with silica gel of a first normal phase silica gel column or not; 
 loading the mixture on the first normal phase silica gel column; 
 gradient eluting the silica gel column with a normal phase silica gel column eluent; 
 collecting fractions containing epothilone B and epothilone A; 
 combining the fractions containing epothilone B and epothilone A; 
 concentrating the combined fractions followed by crystallization to obtain a crude crystal containing epothilones B and A; 
 dissolving the crude crystal containing epothilones B and A in C 1 -C 7  alkyl halide compound(s) to form a second mixture, wherein the second mixture is mixed with silica gel of a second normal phase silica gel column or not; 
 loading the second mixture on a second normal phase silica gel column; 
 gradient eluting the silica gel column with a normal phase silica gel column eluent; 
 collecting fractions containing epothilone B and fractions containing epothilone A respectively; 
 concentrating fractions containing epothilone B and fractions containing epothilone A respectively and crystallizing fractions containing epothilone B. 
 
     
     
       3. The method according to  claim 1 , wherein the C 1 -C 7  alkyl halide compound(s) is selected from one of dichloromethane, trichloromethane and a combination thereof. 
     
     
       4. The method according to  claim 2 , wherein the eluent of the first normal phase silica gel column is a combination of petroleum ether and acetone in a volume ratio of 3-9:1, or a combination of petroleum ether and ethyl acetate in a volume ratio of 1-9:1. 
     
     
       5. The method according to  claim 4 , wherein the eluent of the second normal phase silica gel column is a combination of acetone and petroleum ether in a volume ratio of 1:3-9, or a combination of petroleum ether and acetone in a volume ratio of 1:3-9 with either dichloromethane or trichloromethane and the volume of dichloromethane or trichloromethane is 5%-50% of the total volume. 
     
     
       6. The method according to  claim 5 , wherein the fractions containing epothilones B and/or A eluted from the silica gel columns are measured by HPLC. 
     
     
       7. The method according to  claim 6 , wherein a solvent used for crystallization is n-heptane, ethyl acetate or a combination thereof. 
     
     
       8. The method according to  claim 7 , wherein the solvent used for crystallization is a combination of n-heptane and ethyl acetate having a volume ratio of 1:1. 
     
     
       9. The method according to  claim 8 , wherein crystallization is performed by dissolving a crude product containing epothilones B and A, or a crude product containing epothiloine B in an appropriate amount of ethyl acetate, adding n-heptane therein, letting obtained solution stand at room temperature, then cooling to 4° C. to obtain crystals. 
     
     
       10. The method according to  claim 1 , wherein the sample containing epothilones B and A is a product separated from a fermentation broth of myxobacteria via non-polar macroporous polymeric adsorbents. 
     
     
       11. The method according to  claim 10 , wherein the sample containing epothilones B and A is prepared by a process comprising:
 adding a resin of a first non-polar macroporous polymeric adsorbent column into the fermentation broth of myxobacteria, filtering by a vibrating screen and washing by water to remove impurities at the same time, then loading the resin in a column, gradient eluting with an alcohol solution, and combining fractions containing epothilones B and A; 
 diluting the combined fractions containing epothilones B and A to an appropriate concentration, then loading the diluted solution on a second non-polar macroporous polymeric adsorbent column, gradient eluting with an alcohol solution, collecting fractions containing epothilones B and A, combining the fractions containing epothilones B and A and then obtaining the sample containing epothilones B and A. 
 
     
     
       12. The method according to  claim 11 , wherein the first non-polar macroporous polymeric absorbent is XAD-1600 type resin; and the second non-polar macroporous polymeric absorbent is H41 type resin. 
     
     
       13. The method according to  claim 12 , wherein an eluent used for the first non-polar macroporous polymeric adsorbent column is an ethanol solution of 30%400% by volume; and an eluent used for the second non-polar macroporous polymeric adsorbent column is an ethanol solution of 30%-80% by volume. 
     
     
       14. The method according to  claim 13 , wherein the fractions containing epothilone B and A eluted from the first and the second non-polar macroporous polymeric absorbent columns are measured by HPLC. 
     
     
       15. The method according to  claim 14 , wherein the separation and purification of epothilone B and epothilone A are performed by chromatography of the normal phase silica gel column, the method comprises:
 (1) filtering the fermentation broth wherein XAD-1600 type resin is added by a vibrating screen and washing by water to remove impurities at the same time, 
 then loading the resin in a column, 
 gradient eluting with an ethanol solution of 30%-100% by volume, 
 collecting fractions sectionally, 
 collecting respectively fractions containing epothilone B and epothilone A after analysis by HPLC, and 
 combining fractions containing epothilone B and epothilone A; 
 (2) diluting combined fractions containing epothilones B and A to form an alcohol solution with an appropriate concentration, or concentrating combined fractions to a suitable volume by vacuum evaporation and then diluting to form an alcohol solution with an appropriate concentration, 
 loading the alcohol solution on H41 type resin column, 
 gradient eluting with an alcohol solution of 30%-80% by volume, 
 collecting fractions sectionally, 
 collecting fractions containing epothilone B and epothilone A after analysis by HPLC, 
 combining fractions containing epothilone B and epothilone A, 
 concentrating the combined fractions by vacuum evaporation until dry to obtain a sample containing epothilones B and A; 
 (3) dissolving the sample containing epothilones B and A in trichloromethane or dichloromethane, wherein the mixture is mixed with silica gel of a first normal phase silica gel column or not; 
 loading the mixture on the first normal silica gel column, 
 gradient eluting by a mixture of petroleum ether/acetone or a mixture of petroleum ether/ethyl acetate, 
 collecting fractions sectionally, 
 collecting fractions containing epothilone B and epothilone A after analysis by HPLC, 
 combining fractions containing epothilone B and epothilone A, 
 concentrating the combined fractions by vacuum evaporation until dry, 
 performing crystallization by a mixed solvent of ethyl acetate/n-heptane to obtain crude crystal containing epothilones B and A; 
 (4) dissolving the crude crystal containing epothilones B and A in trichloromethane or dichloromethane, wherein the second mixture is mixed with silica gel of a second normal phase silica gel column or not; 
 loading the second mixture on the second normal silica gel column, 
 gradient eluting by a mixture of petroleum ether/acetone or a mixture of petroleum ether/acetone/trichloromethane, 
 collecting fractions sectionally, 
 collecting respectively fractions containing epothilone B and fractions containing epothilone A after analysis by HPLC; 
 (5) performing crystallization on fractions containing epothilone B by ethyl acetate/n-heptane, 
 dissolving the crystal containing epothilone B in t-butanol and lyophilizing the solution to obtain a product of epothilone B with a high purity in a form of amorphous power; 
 
       dissolving epothilone A in t-butanol and then lyophilizing to obtain a product of epothilone A with a high purity in a form of amorphous power. 
     
     
       16. A method for separation and purification of epothilones B and A, characterized in that, the separation and purification of epothilones B and A are performed by a normal phase silica gel column chromatography, and the method comprises:
 dissolving a sample containing epothilones B and A in C 1 -C 7  alkyl halide compound(s) to form a mixture, wherein the mixture is mixed with silica gel or not; 
 loading the mixture on a normal phase silica gel column; 
 gradient eluting the silica gel column with a normal phase silica gel column eluent; 
 collecting fractions; and 
 obtaining products; 
 
       wherein the normal phase silica gel column is balanced with the C 1 -C 7  alkyl halide compound(s) before use. 
     
     
       17. A method according to  claim 1  wherein a mass ratio of the silica gel used in the first normal phase silica gel column to the sample is 5-10:1; and a mass ratio of the silica gel used in the second normal phase silica gel column to a crude crystal containing epothilones B and A is 50-200:1. 
     
     
       18. A method according to  claim 16  wherein a mass ratio of the silica gel used in the first normal phase silica gel column to the sample is 5-10:1; and a mass ratio of the silica gel used in the second normal phase silica gel column to a crude crystal containing epothilones B and A is 50-200:1. 
     
     
       19. A method according to  claim 16 , wherein the normal phase silica gel column eluent is chosen from a C 1 -C 7  hydrocarbon, a C 1 -C 7  alkyl halide compound, a C 1 -C 7  ketone, a C 1 -C 7  ester, or any combination thereof, and wherein the C 1 -C 7  hydrocarbon is chosen from petroleum ether, n-hexane, cyclohexane, n-heptane, or any combination thereof, the C 1 -C 7  alkyl halide compound is chosen from dichloromethane, trichloromethane, bromoethane, or any combination thereof, the C 1 -C 7  ketone is chosen from acetone, butanone, or any combination thereof, and the C 1 -C 7  ester is chosen from ethyl acetate, isobutyl acetate, or any combination thereof. 
     
     
       20. The method according to  claim 19 , wherein the normal phase silica gel column eluent is chosen from petroleum ether, ethyl acetate, acetone, trichloromethane, dichloromethane, or any combination thereof. 
     
     
       21. The method according to  claim 1 , wherein the normal phase silica gel column eluent is chosen from petroleum ether, ethyl acetate, acetone, trichloromethane, dichloromethane, or any combination thereof.